Alcohol Dehydrogenase Mutant and Application thereof in Synthesis of Diaryl Chiral Alcohols

ABSTRACT

The present disclosure discloses an alcohol dehydrogenase mutant and application thereof in synthesis of diaryl chiral alcohols, and belongs to the technical field of bioengineering. The alcohol dehydrogenase mutant of the present disclosure has excellent catalytic activity and stereoselectivity, and may efficiently catalyze the preparation of a series of chiral diaryl alcohols in R- and S-configurations. By coupling alcohol dehydrogenase of the present disclosure to glucose dehydrogenase or formate dehydrogenase, the synthesis of chiral diaryl alcohol intermediates of various antihistamines may be achieved. Compared with the prior art, a method for preparing diaryl chiral alcohols through asymmetric catalytic reduction using the alcohol dehydrogenase of the present disclosure has the advantages of simple and convenient operation, high substrate concentration, complete reaction and high product purity, and has great industrial application prospects.

TECHNICAL FIELD

The present disclosure relates to an alcohol dehydrogenase mutant andapplication thereof in synthesis of diaryl chiral alcohols, and belongsto the technical field of bioengineering.

BACKGROUND

Chiral diaryl alcohol compounds are key chiral intermediates for thesynthesis of numerous drugs and fine chemicals, where chiral(4-chlorophenyl)-(pyridin-2-yl)-methanol (CPMA) is a key chiralintermediate for the synthesis of an antihistamine drug betahistine. Thesynthesis of chiral CPMA by chemical asymmetric reduction usingprochiral (4-chlorophenyl)-(pyridin-2-yl)-methanone (CPMK) as a rawmaterial is mainly achieved by the following five techniques:

1. at a substrate concentration of 1.0 mM, usingtrans-RuCl₂[(R)-xylbinap][(R)-daipen] as a catalyst to react at roomtemperature for 24 h under the nitrogen pressure of 40-60 psi, so as toobtain (S)-(4-chlorophenyl)-(pyridin-2-yl)-methanol ((S)-CPMA) with anee value of 60.6% and a yield of 98% through reduction. (C. Y. Chen, etal., Org. Lett., 2003, 5, 5039-5042);

2. using (S)-[Ru(BINAP)Cl₂]₂(NE₃) as a catalyst to obtain (S)-CPMA withan ee value of 99% through pressurization, hydrogenation and reduction(Zhao Zhiquan, et al., Chinese Journal of Pharmaceuticals, 2006, 37,726-727);

3. using CPMK as a raw material and (S,S)-6-CHOONa as a catalyst toreact at 50° C. and a substrate concentration of only 0.2 mM for 24 h,so as to obtain (R)-(4-chlorphenyl)-(pyridin-2-yl)-methanol ((R)-CPMA)with an ee value of 40.8% and a yield of 90% through reduction (B. G.Wang, Org. Lett., 2017, 19, 2094-2097);

4. using CPMK as a raw material for three-step reaction, (1) firstprotecting with trifluoromethanesulfonic anhydride and the like, (2)using a catalyst palladium ligand, Me-CBS and the like to reduce acarbonyl group to an S configuration hydroxyl group, and (3) performingdeprotection by triphenylphosphine palladium, so as to obtain (S)-CPMA(Chinese patent CN101848893A); and

5. using chiral BINAL-H as a chiral reducing agent for orientedsynthesis of a single configuration of CPMA at a substrate concentrationof 400 mM CMPK, where after recrystallization of ethyl acetate-petroleumether, the yield of (R)-CPMA is 88.2%, the purity is 96.2%, the yield of(S)-CPMA is 87.4%, and the purity is 95.7% (Chinese patentCN103121966A).

It can be seen that the above reactions have the problems of high costof the noble metal ligand catalysts, low substrate concentration, highpressure conditions for the reactions, many operation steps, and lowoptical purity of the materials, which cannot meet the requirements ofdrugs on the optical purity, and is not favorable for industrialproduction.

Biocatalysis refers to a process of chemical conversion using enzymes orbiological organisms (cells, organelles, tissues, etc.) as a catalystunder mild action conditions, which is completed in an environment ofnormal temperature, a neutral environment, water or the like, and hasunique advantages for the synthesis of chiral active pharmaceuticalingredients. It meets the goals of industrial development such as“sustainable development”, “green chemistry” and “environmentally benignmanufacturing”. Compared with chemical synthesis methods, the use ofalcohol dehydrogenase to asymmetrically reduce the carbonyl group inprochiral ketone has the advantages of high stereoselectivity, mildreaction conditions and the like, and has important economic and socialvalues and ecological significance. The biological asymmetric reductionmethod may be realized mainly by the following four techniques:

1. in 2007, after Truppo et al. screened a series of commercialketoreductases KRED, it was found that although some ketoreductases hada reducing ability to diaryl substrates, the stereoselectivity was justordinary, a substrate spectrum was narrow, and substituent groups in thesubstrates had a great impact on the stereoselectivity; and only KRED124may asymmetrically reduce CPMK to generate (R)-CPMA, the ee value was94%, the conversion rate was 98%, and the addition of glucosedehydrogenase was required to achieve coenzyme circulation (M. D.Truppo, Org. Lett., 2007, 9, 335-338);

2. in 2009, Zhu Dunming et al. discovered that a recombinant carbonylreductase SsCR derived from Sporobolomyces salmonicolor and mutantsthereof may stereoselectively reduce different diaryl ketone substrates(8-99% ee), with the aid of glucose dehydrogenase, (R)-CPMA wasgenerated by reducing CPMK, the conversion rate was 62%, and theenantioselectivity was 88% (R) (D. M. Zhu, Org. Lett., 2008, 10,525-528);

3. in 2012, Zhou Jieyu et al. screened a Kluyveromyces sp. CCTCCM2011385by traditional enrichment culture, which may catalyze the reduction ofCPMK to generate (S)-CPMA (87% ee), however, due to the low content ofactive enzyme in wild fungi, only a 2 g/L substrate may be catalyzed atmost, the product concentration is low, and the separation cost is high,so it cannot meet application needs, (Y. Ni, Process Biochem., 2012, 47,1042-1048; Chinese patent CN102559520A); and

4. in 2013, Li Zhe et al. studied the asymmetric reduction to a seriesof diaryl ketones by a carbonyl reductase PasCR derived from Pichiapastoris GS115, the substrate concentration was 10 mM, and theconversion rate was only 50% at most (LiZhe et al., Chinese Journal ofBiotechnology, 2013, 29, 68-77).

It can be seen that the stereoselectivity for preparing chiral CPMA bythe biological asymmetric reduction method can hardly meet thepharmaceutical requirement for an enantiomeric excess of more than 95%,and in particular, a reductase for synthesizing and preparing (S)-CPMAis unavailable, so there is an urgent need to develop a highly efficientand highly stereoselective bioenzyme catalyst.

SUMMARY

In view of the problem of low stereoselectivity of alcohol dehydrogenasein the prior art, the present disclosure provides a series of alcoholdehydrogenase mutant proteins, a nucleic acid sequence encoding themutant proteins, a recombinant expression vector and a recombinantexpression transformant containing the nucleic acid sequence, andapplication of the alcohol dehydrogenase mutant proteins or therecombinant transformant expressing the alcohol dehydrogenase mutantproteins as a catalyst in asymmetric reduction and preparation of anoptical chiral diaryl alcohol.

The present disclosure provides an alcohol dehydrogenase mutant withhigher reactivity and stereoselectivity.

In an embodiment of the present disclosure, the amino acid sequence ofthe mutant includes an amino acid sequence obtained by mutation of oneor more sites of amino acid glutamine at position 136, amino acidphenylalanine at position 161, amino acid serine at position 196, aminoacid glutamic acid at position 214, amino acid threonine at position 215and amino acid serine at position 237 in the amino acid sequence shownin SEQ ID No. 2.

In an embodiment of the present disclosure, the mutant includes thesubstitution of glycine for glutamic acid at position 214 of the alcoholdehydrogenase with the amino acid sequence shown in SEQ ID No. 2, whichis named M1.

In an embodiment of the present disclosure, the mutant includes thesubstitution of valine for glutamic acid at position 214 of the alcoholdehydrogenase with the amino acid sequence shown in SEQ ID No. 2, whichis named M2.

In an embodiment of the present disclosure, the mutant includes thesubstitution of glycine for glutamic acid at position 214 of the alcoholdehydrogenase with the amino acid sequence shown in SEQ ID No. 2, andthe substitution of cysteine for serine at position 237, which is namedM3.

In an embodiment of the present disclosure, the mutant includes thesubstitution of glycine for glutamic acid at position 214 of the aminoacid sequence shown in SEQ ID No. 2, the substitution of cysteine forserine at position 237, and the substitution of asparaginate forglutamine at position 136, which is named M4.

In an embodiment of the present disclosure, the mutant includes thesubstitution of glycine for glutamic acid at position 214 of the aminoacid sequence shown in SEQ ID No. 2, the substitution of cysteine forserine at position 237, the substitution of asparaginate for glutamineat position 136, and the substitution of glycine for serine at position196, which is named M5.

In an embodiment of the present disclosure, the mutant includes thesubstitution of glycine for glutamic acid at position 214 of the aminoacid sequence shown in SEQ ID No. 2, the substitution of cysteine forserine at position 237, the substitution of asparaginate for glutamineat position 136, the substitution of glycine for serine at position 196,and the substitution of valine for phenylalanine at position 161, whichis named M6.

In an embodiment of the present disclosure, the mutant includes thesubstitution of valine for glutamic acid at position 214 of the aminoacid sequence shown in SEQ ID No. 2, and the substitution of serine forthreonine at position 215, which is named M7.

In an embodiment of the present disclosure, the mutant includes thesubstitution of asparaginate for glutamine at position 136 of alcoholdehydrogenase with an amino acid sequence shown in SEQ ID No. 1, and thesubstitution of valine for phenylalanine at position 161, which is namedM8.

In an embodiment of the present disclosure, the mutant includes thesubstitution of glycine for serine at position 196 of the alcoholdehydrogenase with the amino acid sequence shown in SEQ ID No. 1, andthe substitution of cysteine for serine at position 237, which is namedM9.

In an embodiment of the present disclosure, a recombinant strainexpressing the mutant is provided.

In an embodiment of the present disclosure, a method for constructingthe recombinant strain includes the following steps: cloning anucleotide molecule encoding the mutant into a recombinant vector,transforming the resulting recombinant vector into a transformant toobtain a recombinant expression transformant, and culturing theresulting recombinant expression transformant and conducting isolationand purification to obtain the mutant.

In an embodiment of the present disclosure, the host of the recombinantstrain is Escherichia coli and plasmid is pET28a (+).

In an embodiment of the present disclosure, the host of the recombinantstrain is E. coli BL21 (DE3).

The present disclosure also provides a method for producing an alcoholdehydrogenase by using the recombinant strain, specifically includingthe following steps: inoculating the recombinant strain into an LBmedium containing 40-60 μg/mL kanamycin sulfate for shake cultivation at30-40° C. and 100-200 rpm, adding 0.05-1.0 mMisopropyl-β-D-thiogalactofuranoside (IPTG) for induction at an inducingtemperature of 16-30° C. when the absorbance OD₆₀₀ of a medium solutionreaches 0.5-1.0, and inducing for 5-10 h to obtain the mutant forefficient expression of a recombinant alcohol dehydrogenase.

In an embodiment of the present disclosure, application of the mutant asa catalyst in the preparation of an optical pure chiral diaryl alcoholby asymmetric reduction of a prochiral carbonyl compound is provided.

In an embodiment of the present disclosure, the prochiral carbonylcompound is (4-chlorophenyl)-(pyridin-2-yl)-methanone,phenyl-(pyridin-2-yl)-methanone, (4-chlorophenyl)-(phenyl)-methanone,(4-fluorophenyl-(phenyl)-methanone, (4-bromophenyl)-(phenyl)-methanone,(4-methoxyphenyl)-(phenyl)-methanone, acetophenone, 4-chloroacetophenoneor 4-chlorobenzoyl chloride.

A method for producing chiral CPMA using an alcohol dehydrogenasespecifically includes the following steps: constructing a reactionsystem, where CPMK concentration is 10-500 mM, the amount of thedehydrogenase mutant according to any one of claims 1-3 is 1-10 kU/L,and the amount of NADP+ is 0.1-1.0 mM; adding a coenzyme circulationsystem, wherein the coenzyme circulation system contains glucosedehydrogenase GDH and D-glucose, the amount of glucose dehydrogenase GDHis 1-10 kU/L, the amount of D-glucose dosage is 20-1000 mM, and theconcentration of a phosphate buffer is 0.1-0.2 M; performing reaction at30-35° C. and pH 6-8 for 1-24 h; and extracting the chiral CPMA from areaction solution according to an organic solvent extraction methodafter asymmetric reduction reaction.

In an embodiment of the present disclosure, the coenzyme circulationsystem may also be phosphite/phosphite dehydrogenase (FTDH), formicacid/formate dehydrogenase (FDH), lactic acid/lactate dehydrogenase(LDH) or glycerol/glycerol dehydrogenase.

In an embodiment of the present disclosure, the (R)- and (S)-CPMA ischromatographed by taking 100 μL reaction solution, adding 500 μL ethylacetate, shaking for 1-2 min, centrifuging at 12,000 rpm for 2-5 min,placing a supernatant into a centrifuge tube, and after an organic phaseis naturally volatilized completely, adding 500 μL chromatographic pureethanol for chiral liquid chromatography of a conversion rate and an eevalue. The specific chromatographic conditions are as follows: DaicelChiralcel OB-H (5 μm, 250 mm×4.6 mm) liquid chromatography column,mobile phases are n-hexane: ethanol: ethanolamine (90:10:0.01, v/v/v),the flow rate is I mL/min, the column temperature is 30° C., the UVdetection wavelength is 254 nm, the injection volume is 10 μL, and theretention time for (R)-CPMA and that for (S)-CPMA are 11.14 min and12.34 min respectively.

The present disclosure has the beneficial effects that:

(1) the alcohol dehydrogenase mutant obtained in the present disclosurehas high activity to various carbonyl compounds, and may catalyze thereduction of a plurality of aliphatic or aryl-substituted ketonesubstrates, especially diaryl ketone substrates having a large sterichindrance, and molecular modification on KpADH is achieved through thecombination of mutation means to increase the stereoselectivity of theenzyme, which will make it more industrially useful;

(2) compared with the wild type alcohol dehydrogenase KpADH, the alcoholdehydrogenase mutant M6 of the present disclosure has an invertedS-stereoselectivity for the substrate CPMK, the ee value of a productCPMA is reversed to 97.8% (S) from 82% (R) of the wild type, M7 has ahigher R-stereoselectivity for the substrate CPMK, and the ee value of aproduct CPMA is increased to 99% (R) or above from 82% (R) of the wildtype. The alcohol dehydrogenase mutant obtained in the presentdisclosure is particularly suitable for asymmetric reduction of diarylketones, and has good industrial application prospects.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 is a whole plasmid PCR nucleic acid electrophoretogram of wildtype and alcohol dehydrogenase mutants M1 to M7.

FIG. 2 is SDS-PAGE analysis of alcohol dehydrogenase mutants M1 to M7,respectively.

FIG. 3 is a chiral liquid chromatogram of a product produced from CPMKreduction catalyzed by an alcohol dehydrogenase mutant M6.

FIG. 4 is a chiral liquid chromatogram of a product produced from CPMKreduction catalyzed by an alcohol dehydrogenase mutant M7.

DETAILED DESCRIPTION

The present disclosure will be described in detail below by means ofspecific Examples, but this does not limit the present disclosure to thescope of the described Examples. The experimental methods withoutindicated specific experimental conditions in the following Examples maybe selected according to conventional methods and conditions, oraccording to the specification.

Example 1: Method for Measuring Activity of Alcohol Dehydrogenase

Adopting a total reaction system of 200 μL including: 1.0 mM NADPH, 1.0mM substrate CPMK and sodium phosphate buffer (PBS, 100 mM, pH 7.0),fully and evenly mixing, maintaining at 30° C. for 2 min, adding anappropriate amount of enzyme solution, and detecting the change in lightabsorption at 340 nm.

The enzyme activity was calculated by the following formula:

Enzyme activity (U)=EW×V×10³/(6220×1);

in the formula, EW is the change in absorbance at 340 nm in 1 min; V isthe volume of a reaction solution in mL; 6220 is the molar extinctioncoefficient of NADPH in L/(mol·cm); and 1 is the optical path distancein cm. One activity unit (U) corresponds to the amount of enzymerequired to catalyze the oxidation of 1 μmol NADPH per minute under theabove conditions.

Method for determining optical purity ee:

${{ee} = {\frac{{AS} - {AR}}{{AS} + {AR}} \times 100\%}};$

As: molar concentration of (S)-CPMA obtained by liquid chromatography;and A_(R): molar concentration of (R)-CPMA obtained by liquidchromatography.

Example 2: Construction of Alcohol Dehydrogenase Mutant Gene andRecombinant Expression Transformant

A whole plasmid PCR method was used for site-directed mutagenesis onamino acid residues at positions 136, 161, 196, 214, 215 and 237 toconstruct an iterative combination mutant. The primer design was asfollows (all described in the 5′-3′ direction, and the underlinerepresents the mutation site):

M1 (using pET28a-KpADH recombinant plasmid as a template):

E214G-F: (SEQ ID NO. 3) AAG AAACTA AAT GGTACT TGT; E214G-R:(SEQ ID NO. 4) AAT TTCACA AGTACC ATT TAG;

M2 (using pET28a-KpADH recombinant plasmid as a template):

E214V-F: (SEQ ID NO. 5) AAG AAACTA AATGTTACT TGT; E214V-R:(SEQ ID NO. 6) AAT TTCACA AGTAAC ATT TAG;

M3 (using M1 recombinant plasmid as a template):

S237C-F: (SEQ ID NO. 7) AAGACTCACTTCTGTCAATTC; S237C-R: (SEQ ID NO. 8)ATCAATGAATTGACAGAAGTG;

M4 (using M3 recombinant plasmid as a template):

Q136N-F: (SEQ ID NO. 9) ACCCCACATAGAAATAATGAT; Q136N-R: (SEQ ID NO. 10)AGTTGGATCATTATTTCTATG;

M5 (using M4 recombinant plasmid as a template):

S196G-F: (SEQ ID NO. 11) ACTATCCACCCAGGTTTCGTT; S196G-R: (SEQ ID NO. 12)TCCGAAAACGAAACCTGGGTG;

M6 (using M5 recombinant plasmid as a template):

F161V-F: (SEQ ID NO. 13) TATGAAAATGTCGTTACTGCT; F161V-R: (SEQ ID NO. 14)ACAATAAGCAGTAACGACATT;

M7 (using pET28a-KpADH recombinant plasmid as a template):

E214V/T215S-F: (SEQ ID NO. 15) AAGAAACTAAATGTTAGCTGTGAA; E214V/T215S-R:(SEQ ID NO. 1+) GATAATTTCACAGCTAACATTTAG;

M8 (using pET28a-KpADH_(Q136N) recombinant plasmid as a template):

F161V-F: (SEQ ID NO. 17) TATGAAAATGTCGTTACTGCT; F161V-R: (SEQ ID NO. 18)ACAATAAGCAGTAACGACATT;

M9 (using pET28a-KpADH_(S196G) recombinant plasmid as a template):

S237C-F: (SEQ ID NO. 19) AAGACTCACTTCTGTCAATTC; S237C-R: (SEQ ID NO. 20)ATCAATGAATTGACAGAAGTG.

A PCR reaction system was: a PCR reaction system (50 μL) including KODenzyme (2.5 U/mL) 1.0 μL, template (5-50 ng) 1.0 μL, dNTP 4.0 μL, 10×reaction buffer 5.0 μL, forward primer 1.0 μL, reverse primer 1.0 μL,and the rest of ddH2O to make the reaction system 50 μL in total.

A PCR amplification procedure was: (1) denaturation at 94° C. for 3 min,(2) denaturation at 94° C. for 30 sec, (3) annealing at 54° C. for 30sec, (4) extension at 72° C. for 150 sec, repeating steps (2) to (4) for10-15 cycles, finally extension at 72° C. for 10 min, and storing a PCRamplification product at 4° C.

After PCR, DpnI restriction enzyme was added into a reaction mixture andincubated at 37° C. for 1 h, 10 μL digested PCR reaction solution wastransferred into 50 μL E. coli BL21 (DE3) competent cells through CaCl₂thermal transformation, and the cells were used to uniformly coat an LBagar plate containing 50 μg/mL kanamycin sulfate for inversion cultureat 37° C. for 12 h.

Example 3: Expression and Purification of Alcohol Dehydrogenase andMutant Thereof

Recombinant Escherichia coli carrying a stereoselective improvementmutant was inoculated into an LB medium containing kanamycin sulfate (50μg/mL) at a transfer amount of 2% for shake cultivation at 37° C. and200 rpm, 0.2 mM isopropyl-β-D-thiogalactofuranoside (IPTG) was added forinduction at 25° C. when the absorbance OD₆₀₀ of the medium reached 0.8,after 8 hours of induction, a strain for efficient expression of arecombinant alcohol dehydrogenase mutant was obtained through 10 minutesof centrifugation at 8000 rpm, and the collected strain was suspended ina potassium phosphate buffer (100 mM, pH 6.0) for ultrasonication.

A column used for purification was a nickel affinity column HisTrap FFcrude, and purification was achieved through affinity chromatographyusing a histidine tag on recombinant protein. The nickel column wasequilibrated with a solution A first, a crude enzyme solution wasloaded, a penetrating peak was further eluted off using the solution A,and after equilibrium, a solution B (20 mM sodium phosphate, 500 mMNaCl, and 1000 mM imidazole, pH 7.4) was used for gradient elution toelute off the recombinant protein bound to the nickel column, so as toobtain the recombinant alcohol dehydrogenase mutant. The purifiedprotein was subjected to activity measurement (CPMK as substrate, andNADPH as coenzyme) and SDS-PAGE analysis. After purification of thenickel column, a single band was displayed at around 45 kDa, and theamount of impure protein was small, indicating that the columnpurification effect was good. The purified alcohol dehydrogenase proteinwas then replaced into a Tris-HCl (100 mM, pH 7.0) buffer using a HiTrap Desalting column (GE Healthcare).

Example 4: Kinetic and Stereoselective Analysis of Alcohol DehydrogenaseMutant

The activity of KpADH at different substrate concentrations and coenzymeconcentrations was determined, and a double reciprocal curve was madebased on the reciprocal of activity and substrate concentration tocalculate kinetic parameters.

It can be seen from Table 1 that the k_(cat)/K_(m) of KpADH to CPMK was28.9 s⁻¹·mM⁻¹, the reduction product configuration was R configuration,and the ee value was 82.5%. The stereoselectivity of (R)-CPMAsynthesized by mutants M2 and M7 was increased, and the ee values of theproducts were 92.3% and 99.1% respectively. Mutant M1 showed a reducedstereoselectivity, the reduction product configuration was also Rconfiguration, and the ee value of the product was 3.29%. Mutants M3,M4, M5 and M6 showed an inverted stereoselectivity, the reductionproducts were in the S configuration, and the ee values of the productswere 51.8%, 88.0%, 93.5% and 97.8% respectively. The reduction productsof the control examples M8 and M9 were in the R configuration, the eevalues of the products were little different from that of the wild typeKpADH, and the mutation had no effect on the stereoselectivity of theenzyme.

TABLE 1 Kinetic parameters and stereoselectivity of alcoholdehydrogenase mutant Vmax Kcat/Km Km (μmol · min⁻¹ · Kcat (s⁻¹ · eeConfig. Enzyme (mM) mg⁻¹) (s⁻¹) mM⁻¹) (%) (R/S) KpADH 0.410 17.9 11.828.9 82.5 R M1 0.52 11.1 7.32 14.1 3.29 R M2 0.574 17.5 11.7 14.2 92.3 RM3 0.632 9.32 6.16 9.74 51.8 S M4 0.861 10.2 6.76 7.85 88.0 S M5 0.7221.3 17.0 23.6 93.5 S M6 1.12 25.2 20.1 17.9 99.8 S M7 0.702 21.3 14.220.3 99.9 R M8 (control 0.604 22.3 14.8 24.6 83.5 R example) M9 (control0.730 26.5 17.6 24.1 81.7 R example)

Example 5: Substrate Specificity Analysis of Alcohol DehydrogenaseMutant

The reduction on prochiral carbonyl compounds by the alcoholdehydrogenase mutants obtained in Example 2 was studied, and the studiedprochiral carbonyl compounds include(4-chlorophenyl)-(pyridin-2-yl)-methanone (CPMK),phenyl-(pyridin-2-yl)-methanone, (4-chlorophenyl)-(phenyl)-methanone,(4-bromophenyl)-(phenyl)-methanone,(4-fluorophenyl)-(pyridin-2-yl)-methanone,(4-methoxyphenyl)-(phenyl)-methanone, acetophenone, 4-chloroacetophenoneand 2-(4-chlorophenyl)acetyl chloride.

TABLE 2 Substrate specificity of alcohol dehydrogenase mutant M8 M9Substrate WT M1 M2 M3 M4 M5 M6 M7 (control) (control)

81.9 (R) 24.2 (R) 92.3 (R) 65.4 (S) 80.6 (S) 87.7 (S) 99.8 (S) 99.9 (R)83.5 (R) 81.7 (R)

26.3 (R) 41.0 (R) 30.6 (R) 36.5 (R) 20.7 (R) 15.5 (R)  6.81 (S) 11.9 (R)22.3 (R) 42.3 (R)

71.4 (S) 63.2 (S) 87.9 (S) 33.2 (R) 38.6 (R) 62.0 (R) 91.7 (R) 99.4 (S)52.4 (S) 78.4 (S)

69.2 (S) 49.3 (S) 95.5 (S) 63.3 (R) 66.5 (R) 70.9 (R) 95.1 (R) 99.6 (S)70.2 (S) 59.2 (S)

25.3 (R)  7.51 (R)  0.56 (S) 45.6 (R) 49.1 (R) 59.5 (R) 64.2 (R)  9.22(S) 55.3 (R) 67.3 (R)

14.9 (R) 42.7 (R) 23.2 (R) 80.6 (R) 82.6 (R) 81.7 (R) 95.1 (R) 45.7 (S)24.5 (R) 49.1 (R)

59.2 (R) 68.5 (R) 45.3 (R) 95.3 (R) 91.5 (R) 95.6 (R) 95.3 (R) 51.3 (R)66.2 (R) 49.7 (R)

67.1 (R) 86.3 (R) 94.9 (R) 88.9 (R) 57.7 (R) 49.1 (R) 82.1 (R) 95.1 (R)77.1 (R) 47.3 (R)

77.8 (R) 94.5 (R) 91.2 (S) 99.8 (R) 99.3 (R) 99.9 (R) 99.9 (R) 77.5 (R)57.2 (R) 42.8 (R)

As can be seen from Table 2, compared with WT, the combined mutant M6obtained by iterative combination mutation had an inversestereoselectivity for (4-chlorophenyl)-(pyridin-2-yl)-methanone (CPMK),4-bromophenyl-(pyridin-2-yl)-methanone and 4-methoxy-(phenyl)-methanone,and the ee values of products were all 95% or above; the mutant M6 hadthe same stereoselectivity as the wild type for 2-(4-chlorophenyl)acetylchloride, and the ee values of products were all 99% or above; and themutant M7 had the same stereoselectivity as WT for(4-chlorophenyl)-(pyridin-2-yl)-methanone (CPMK), and(4-bromophenyl)-(phenyl)-methanone, and the ee values of products wereover 99%. Experiments have shown that the combination mutant strainsobtained through iterative combination mutation had high R- andS-stereoselectivity for aryl ketone, especially large stericallyhindered diaryl ketone substrates, and may be used as biocatalysts forpreparation of R- and S-configuration chiral aryl alcohol intermediates.

Example 6: Preparation of (S)-CPMA and (R)-CPMA Through AsymmetricReduction of CPMK by Alcohol Dehydrogenase Mutant

A 20 mL biocatalytic system was established: 100 mM potassium phosphatebuffer (pH 7.0), and the alcohol dehydrogenase mutants M6 and M7obtained in Example 2 as well as wild KpADH 10 g/L, CPMK 100 mM, 200 mMand 500 mM were added (substrate added in batches). The reaction wasperformed at 30° C. and 200 rpm for 12 h with a constant pH of 7.5.

The conversion rate analysis results during the reaction are shown inTable 3, Table 4 and Table 5. It can be seen that both the wild typedehydrogenase and the mutants M6 and M7 may asymmetrically reduce 100 mMand 200 mM CPMK. When the CPMK concentration was 200 mM, the wild typeKpADH and the two mutants (M6 and M7) required 12 h and 24 hrespectively to achieve a conversion rate close to 99.9%. The finalreduction product of the wild type KpADH was (R)-CPMA, and the ee valuewas 82%; the final reduction product of the mutant M6 was (S)-CPMA, andthe ee value was 99.5%; and the final reduction product of the mutant M7was (R)-CPMA, and the ee value was 99.7%. The obtained crude products of(R)-CPMA and (S)-CPMA were redissolved in ethanol, and correspondingpure products of (R)-CPMA and (S)-CPMA were added as seed crystals torecrystallize at 4° C. to finally obtain products with optical puritygreater than 99.9% ee.

TABLE 3 Asymmetric reduction of CPMK catalyzed by wild type alcoholdehydrogenase KpADH Conversion rate (%) Reaction time (h) 100 mM 200 mM500 mM 1 47.76 25.6 11.7 2 77.9 36.9 20.1 3 87.1 50.5 44.8 4 96.5 62.859.6 6 98.7 85.3 80.2 8 99.6 97.4 93.2 12 >99.9 99.4 95.6 24 >99.9 99.799.2

TABLE 4 Asymmetric reduction of CPMK catalyzed by alcohol dehydrogenasemutant M6 Conversion rate (%) Reaction time (h) 100 mM 200 mM 500 mM 124.6 19.1 12.8 2 54.6 36.4 22.1 3 69.4 52.8 30.6 4 80.2 70.6 65.3 6 95.289.7 77.9 8 97.2 92.2 87.6 12 98.2 95.4 90.2 24 99.6 99.2 99.5

TABLE 5 Asymmetric reduction of CPMK catalyzed by alcohol dehydrogenasemutant M7 Conversion rate (%) Reaction time (h) 100 mM 200 mM 500 mM 134.0 20.9 10.8 2 40.8 33.1 21.1 3 41.5 48.1 38.7 4 46.5 55.2 43.2 6 58.777.4 62.1 8 69.0 86.3 70.7 12 99.7 92.2 89.9 24 99.7 99.7 99.4

From this, it is understood that the alcohol dehydrogenase mutantenzymes M6 and M7 of the present disclosure have very good performancein terms of efficient, highly stereoselective asymmetric reduction ofCPMK.

What is claimed is:
 1. An alcohol dehydrogenase mutant, wherein an aminoacid sequence of the mutant comprises mutation of one or more amino acidsites in an amino acid sequence shown in SEQ ID NO.
 2. 2. The mutantaccording to claim 1, wherein the mutation comprises mutation of aminoacid glutamine at position 136, mutation of amino acid phenylalanine atposition 161, mutation of amino acid serine at position 196, amino acidglutamic acid at position 214, mutation of amino acid threonine atposition 215, mutation of amino acid serine at position 237, or acombination thereof in the amino acid sequence shown in SEQ ID No.
 2. 3.The mutant according to claim 1, wherein the mutation comprisessubstitution of any one of the following: a substitution of glycine forglutamic acid at position 214 of the amino acid sequence shown in SEQ IDNo. 2; a substitution of valine for glutamic acid at position 214 of theamino acid sequence shown in SEQ ID No. 2; a substitution of glycine forglutamic acid at position 214 of the amino acid sequence shown in SEQ IDNo. 2, and a substitution of cysteine for serine at position 237; a thesubstitution of glycine for glutamic acid at position 214 of the aminoacid sequence shown in SEQ ID No. 2, a substitution of cysteine forserine at position 237, and a substitution of asparaginate for glutamineat position 136; a substitution of glycine for glutamic acid at position214 of the amino acid sequence shown in SEQ ID No. 2, a substitution ofcysteine for serine at position 237, a substitution of asparaginate forglutamine at position 136, and a substitution of glycine for serine atposition 196; a substitution of glycine for glutamic acid at position214 of the amino acid sequence shown in SEQ ID No. 2, a substitution ofcysteine for serine at position 237, a substitution of asparaginate forglutamine at position 136, a substitution of glycine for serine atposition 196, a the substitution of valine for phenylalanine at position161; and a substitution of valine for glutamic acid at position 214 ofthe amino acid sequence shown in SEQ ID No. 2, and a substitution ofserine for threonine at position
 215. 4. The mutant according to claim2, wherein the mutation comprises substitution of any one of thefollowing: a substitution of glycine for glutamic acid at position 214of the amino acid sequence shown in SEQ ID No. 2; a substitution ofvaline for glutamic acid at position 214 of the amino acid sequenceshown in SEQ ID No. 2; a substitution of glycine for glutamic acid atposition 214 of the amino acid sequence shown in SEQ ID No. 2, and asubstitution of cysteine for serine at position 237; a substitution ofglycine for glutamic acid at position 214 of the amino acid sequenceshown in SEQ ID No. 2, a substitution of cysteine for serine at position237, and a substitution of asparaginate for glutamine at position 136; asubstitution of glycine for glutamic acid at position 214 of the aminoacid sequence shown in SEQ ID No. 2, a substitution of cysteine forserine at position 237, a substitution of asparaginate for glutamine atposition 136, and a substitution of glycine for serine at position 196;a substitution of glycine for glutamic acid at position 214 of the aminoacid sequence shown in SEQ ID No. 2, a substitution of cysteine forserine at position 237, a substitution of asparaginate for glutamine atposition 136, a substitution of glycine for serine at position 196, anda substitution of valine for phenylalanine at position 161; and asubstitution of valine for glutamic acid at position 214 of the aminoacid sequence shown in SEQ ID No. 2, and a substitution of serine forthreonine at position
 215. 5. A nucleotide sequence encoding the mutantaccording to claim
 1. 6. A nucleotide sequence encoding the mutantaccording to claim
 2. 7. A nucleotide sequence encoding the mutantaccording to claim
 3. 8. A recombinant strain expressing the mutantaccording to claim
 1. 9. A recombinant strain expressing the mutantaccording to claim
 2. 10. A recombinant strain expressing the mutantaccording to claim
 3. 11. A method for constructing the recombinantstrain according to claim 8, wherein the method specifically comprisesthe following steps: cloning a nucleotide sequence encoding the mutantinto a recombinant vector, transforming the resulting recombinant vectorinto a host to obtain a recombinant transformant, and culturing therecombinant transformant and conducting isolation and purification toobtain the mutant.
 12. The method according to claim 11, wherein thehost of the recombinant strain is Escherichia coli, and plasmid ispET28a (+).
 13. The method according to claim 11, wherein the host ofthe recombinant strain is E. coli BL21 (DE3).
 14. The method accordingto claim 12, wherein the host of the recombinant strain is E. coli BL21(DE3).
 15. A method for producing an alcohol dehydrogenase mutant byusing the recombinant strain according to claim 8, wherein the methodcomprises: inoculating the recombinant strain into an LB mediumcontaining 40-60 μg/mL kanamycin sulfate for shake cultivation at 30-40°C. and 100-200 rpm, adding 0.05-1.0 mMisopropyl-β-D-thiogalactofuranoside (IPTG) for induction at an inducingtemperature of 16-30° C. when the absorbance OD₆₀₀ of a medium solutionreaches 0.5-1.0, and inducing for 5-10 h to obtain the mutant forefficient expression of a recombinant alcohol dehydrogenase.
 16. Amethod for producing an alcohol dehydrogenase mutant by using therecombinant strain according to claim 9, wherein the method comprises:inoculating the recombinant strain into an LB medium containing 40-60μg/mL kanamycin sulfate for shake cultivation at 30-40° C. and 100-200rpm, adding 0.05-1.0 mM isopropyl-β-D-thiogalactofuranoside (IPTG) forinduction at an inducing temperature of 16-30° C. when the absorbanceOD₆₀₀ of a medium solution reaches 0.5-1.0, and inducing for 5-10 h toobtain the alcohol dehydrogenase mutant.
 17. A method for producing analcohol dehydrogenase mutant by using the recombinant strain accordingto claim 10, wherein the method comprises: inoculating the recombinantstrain into an LB medium containing 40-60 μg/mL kanamycin sulfate forshake cultivation at 30-40° C. and 100-200 rpm, adding 0.05-1.0 mMisopropyl-β-D-thiogalactofuranoside (IPTG) for induction at an inducingtemperature of 16-30° C. when the absorbance OD₆₀₀ of a medium solutionreaches 0.5-1.0, and inducing for 5-10 h to obtain the alcoholdehydrogenase mutant.
 18. A method for producing chiral CPMA using thealcohol dehydrogenase mutant of claim 1, wherein the method comprisesthe following steps: constructing a reaction system, wherein CPMKconcentration is 10-500 mM, an amount of the alcohol dehydrogenasemutant is 1-10 kU/L, and an amount of NADP⁺ is 0.1-1.0 mM; adding acoenzyme circulation system, wherein the coenzyme circulation systemcontains glucose dehydrogenase GDH and D-glucose, wherein an amount ofglucose dehydrogenase GDH is 1-10 kU/L, an amount of D-glucose dosage is20-1000 mM, and a concentration of a phosphate buffer is 0.1-0.2 M;performing reaction at 30-35° C. and pH 6-8 for 1-24 h; and extractingthe chiral CPMA from a reaction solution according to an organic solventextraction method after asymmetric reduction reaction; wherein thecoenzyme circulation system comprises phosphite/phosphite dehydrogenase(FTDH), formic acid/formate dehydrogenase (FDH), lactic acid/lactatedehydrogenase (LDH) or glycerol/glycerol dehydrogenase.
 19. A method forproducing chiral CPMA using the alcohol dehydrogenase mutant of claim 2,wherein the method comprises the following steps: constructing areaction system, wherein CPMK concentration is 10-500 mM, an amount ofthe alcohol dehydrogenase mutant is 1-10 kU/L, and an amount of NADP⁺ is0.1-1.0 mM; adding a coenzyme circulation system, wherein the coenzymecirculation system contains glucose dehydrogenase GDH and D-glucose,wherein an amount of glucose dehydrogenase GDH is 1-10 kU/L, an amountof D-glucose dosage is 20-1000 mM, and a concentration of a phosphatebuffer is 0.1-0.2 M; performing reaction at 30-35° C. and pH 6-8 for1-24 h; and extracting the chiral CPMA from a reaction solutionaccording to an organic solvent extraction method after asymmetricreduction reaction; wherein the coenzyme circulation system comprisesphosphite/phosphite dehydrogenase (FTDH), formic acid/formatedehydrogenase (FDH), lactic acid/lactate dehydrogenase (LDH) orglycerol/glycerol dehydrogenase.
 20. A method for producing chiral CPMAusing the alcohol dehydrogenase mutant of claim 3, wherein the methodspecifically comprises the following steps: constructing a reactionsystem, wherein CPMK concentration is 10-500 mM, an amount of thealcohol dehydrogenase mutant is 1-10 kU/L, and an amount of NADP⁺ is0.1-1.0 mM; adding a coenzyme circulation system, wherein the coenzymecirculation system contains glucose dehydrogenase GDH and D-glucose,wherein an amount of glucose dehydrogenase GDH is 1-10 kU/L, an amountof D-glucose dosage is 20-1000 mM, and a concentration of a phosphatebuffer is 0.1-0.2 M; performing reaction at 30-35° C. and pH 6-8 for1-24 h; and extracting the chiral CPMA from a reaction solutionaccording to an organic solvent extraction method after asymmetricreduction reaction; wherein the coenzyme circulation system comprisesphosphite/phosphite dehydrogenase (FTDH), formic acid/formatedehydrogenase (FDH), lactic acid/lactate dehydrogenase (LDH) orglycerol/glycerol dehydrogenase.